User:Karmella Haynes/Notebook/miR Trigger/2011/07/10

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07/10/11

  • ✓ Transfections: miR-34 sensor



Transfections: miR-34 sensors
> Lipofectamine LTX
> Follow Jason's protocol, 12-well plates

  1. Plate 1: 12-well glass-bottom for time course microscopy
  2. Plate 2: 12-well plastic for KAH183-177/MV8 only control (no miR-34 target sites) for flow cytometry


Plate 1

Wells Plasmid (MV8 vector) </u>
1, 2 183-DF4
3, 4 183-DF5
5, 6 183-DF6
7, 8 183-177 (no miR-34 t.s.)


Lipo LTX
Note: set up 2x vol reactions
> Dilute 1 μg DNA in dH2O (10 μL final); 3x = 2 μg DNA in dH2O (20 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA; 2x = 380 μL Opti-MEM, 20 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.; 2x = 2 μL PLUS
> Add 3 μL Lipo LTX --> R.T/ 30 min.; 2x = 6 μL Lipo LTX
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

> Microscopy tomorrow (7/11/11)


Plate 2

Wells Plasmid (MV8 vector) </u>
1-3 183-177 (no miR-34 t.s.)
4-6 no DNA

Lipo LTX
Note: set up 3x vol reactions
> Dilute 1 μg DNA in dH2O (10 μL final); 3x = 3 μg DNA in dH2O (30 μL final)
> Add 190 μL Opti-MEM to 10 μL DNA; 3x = 570 μL Opti-MEM, 30 μL DNA
> Add 1 μL PLUS reagent --> R.T/ 5 min.; 3x = 3 μL PLUS
> Add 3 μL Lipo LTX --> R.T/ 30 min.; 3x = 9 μL Lipo LTX
> Add ~200 μL complexes (drop-wise) to each well (1 ml med. each); Grow cells at 37°C

> Flow cytometry tomorrow (7/11/11)